Accurate chromosome segregation is crucial to ensure that each daughter cell receives a complete copy of the genetic information. While the molecular mechanisms underpinning this process have been identified in eukaryotes, even forty years after the first model for chromosome segregation in bacteria was proposed in the replicon hypothesis, we know little about these events in prokaryotes. Recent observations from several laboratories indicate that chromosome segregation in E. coli and B. subtilis is not a passive event, but is orchestrated by forces generated by the processes of replication and transcription and managed by DNA packaging proteins. The long range goal of the studies supported by this grant has been to understand the roles in DNA metabolism of the four topoisomerases found in E. coil. During the previous grant period these studies led us to investigate the intersection between topoisomerase IV-catalyzed decatenation of daughter chromosomes, chromosome partition, and chromosome dynamics. We have: i) shown that the activity of Topo IV is temporally regulated in the cell as a result of independent cellular localization of the subunits of the enzyme and that this regulation is required for efficient chromosome decatenation; ii) demonstrated an interaction between Topo IV and the septal ring protein FtsK that stimulates the chromosome decatenation activity of Topo IV, thereby linking topological separation of the sister chromosomes to the cell division apparatus; and iii) uncovered the participation in chromosome segregation of SpcA, an integral inner membrane protein that interacts with MreB, the bacterial actin ancestor, potentially linking chromosome segregation to cellular infrastructure. We will proceed to use a combination of biochemical, cell biologic, and molecular genetic approaches to answer the following questions: What are the molecular mechanisms underlying temporal regulation of Topo IV activity in the cell? What is the role of the Topo IV-FtsK interaction in chromosome segregation? And, what is the role of SpcA in chromosome segregation?